Ternary regulation mechanism of Rhizoma drynariae total flavonoids on induced membrane formation and bone remodeling in Masquelet technique

Context Rhizoma drynariae total flavonoids (RDTF) are used to treat fractures. CD31hiEmcnhi vessels induced by PDGF-BB secreted by osteoclast precursors, together with osteoblasts and osteoclasts, constitute the ternary regulatory mechanism of bone tissue reconstruction. Objective This study aimed to determine whether RDTF can promote bone tissue remodeling and induce membrane growth in the rat Masquelet model and to explore its molecular mechanism based on the ternary regulation theory. Methods Thirty-six SD rats were randomized to three groups: blank, induced membrane, and RDTF treatment (n = 12/group). The gross morphological characteristics of the new bone tissue were observed after 6 weeks. Sixty SD rats were also randomized to five groups: blank, induction membrane, low-dose RDTF, medium-dose RDTF, and high-dose RDTF (n = 12/group). After 4 weeks, immunohistochemistry and western blot were used to detect the expression of membrane tissue-related proteins. The mRNA expression of key factors of ternary regulation was analyzed by qRT-PCR. Results RDTF positively affected angiogenesis and bone tissue reconstruction in the bone defect area. RDTF could upregulate the expression of key factors (PDGF-BB, CD31, and endomucin), VEGF, and HMGB1 mRNA and proteins in the ternary regulation pathway. Discussion and conclusion Although the expected CD31hiEmcnhi vessels in the induction membrane were not observed, this study confirmed that RDTF could promote the secretion of angiogenic factors in the induced membrane. The specific mechanisms still need to be further studied.

In this study we focused on whether RDTF can promote induce membrane growth and bone remodeling in the rat Masquelet model and explore its molecular mechanism based on ternary regulation theory.

Animals and Masquelet's technique model establishment
Masquelet's technique was performed in a rat femur defect model. Totally ninety-six specific pathogen-free (SPF) male rats (8-10 weeks of age, weighing 260-280 g) were provided by the Laboratory Animal Center of Guangzhou University of Traditional Chinese Medicine(ethical number: SCX-2018-0034). All animals were treated in compliance with the principles and procedures contained in the most recent publication of the National Institutes of Health guide for the care and use of laboratory animals. The rats were housed four animals/cage, at 24-26˚C and 60-80% humidity, with 14 h of light/day. They received rat chow and water ad libitum. For the stage one, rats were general intraperitoneal anesthesia (5% chloral hydrate, 8 mg/kg), the right hindlimb was shaved, and the animals were placed in a lateral position. Penicillin (80,000 U, North China Pharmaceutical Co. Ltd, China) was injected intramuscularly before surgery to prevention of infection. A longitudinal incision was made in the skin and superficial fascia over the right femur. The biceps femoris and vastus lateralis muscles were separated and elevated from the greater trochanter, exposing the lateral aspect of the femoral bone. A sixhole, 1.5-mm homemade stainless steel mini-plate was applied to the lateral aspect of the femur shaft. Two 1.5-mm cortical screws (Suzhou Yuelai Medical Equipment Co., Ltd., Jiangsu, China) were drilled in holes #1 and #6 for fixation (S1 Fig). Then, two Kirshner wires were drilled into holes #2 and #5. The osteotomy guide plate was placed above the steel plate after passing through the holes. With the osteotomy guide plate was secured, a critical-sized defect (CSD) measuring 4 mm was created in the femur bone shaft, using a reciprocating saw. The bone defect was filled with PMMA cement and molded into a cylindrical shape. After securing the cement with silk thread, the wound was washed with sterile water, and the fascia and skin were sewn. 4 weeks after the stage one surgery the stage 2 surgery was conducted, preoperative preparation and surgical approach were the same as that of the first stage. The induced membrane was carefully cut open, PMMA cement was removed, and two segments of rat coccyx were taken as bone graft materials to implant in the bone defect area. The induced membrane tissue was sutured, and muscle, deep fascia, and skin were sutured in turn. The animals were returned to their cages and house separated. Penicillin was given for 5 days postoperatively, during which time the rats were monitored daily for the occurrence of abnormal behavior and/or complications. Meloxicam (0.2mg/kg) in the first 3 days after operative to analgesia. Under general anesthesia, the membranes surrounding PMMA cement spacer were harvested 4 weeks after the first stage. After the experiment, the animals were euthanized with an overdose (150 mg/kg) of pentobarbital administered intraperitoneally.

Treatment
Experiment 1, 60 rats were randomly divided into five groups (empty control group, EG; induced membrane group, IG; low dose RDTF group, LG; medium dose group, MG; high dose group, HG). The operation for the bone defect was performed as above, and no second stage surgery was performed. The EG group was not implanted with PMMA cement.
Approximately 24 h after the animal model was established, the LG, MG, and HG groups were administered with QiangGu capsules (contain 180mg RDTF/each, National Medicine Permit No. Z20030007; batch #180304) intragastrically. Mixed the powder in distilled water, and administered by gavage, the intervention concentrations were 157.5 mg/kg/d for HG, 78.75 mg/kg/d for MG, and 39.38 mg/kg/d for LG. The animals in the EG and IG groups were given the same amount of pure water. Gavage was started on the first day after modeling and was performed once a day until the sampling day of the fourth week after surgery. Histological and radiographs were performed routinely at 4 weeks postoperatively. The rats with complications were not included in the analyses to avoid confounding factors from infections and inflammation. Experiment 2, 36 rats were randomly divided into three groups (empty control group, ECG; induced membrane group, IMG; induced membrane plus total flavonoids group, IFG). The operation process of the bone defect was performed as above. The ECG group was not implanted with PMMA cement. The second stage surgery was performed 4 weeks after PMMA cement implantation. Bone grafting was not performed in the ECG group, and the postoperative treatment was the same as that in the first stage. QiangGu capsules (contain 180mg RDTF/each, National Medicine Permit No. Z20030007; batch #180304) were used. Approximately 24 hours after the animal model was established, the IMG and IFG groups were administered QiangGu capsules mixed in distilled water., the dose was 78.75 mg/kg/d, The ECG and IMG groups were fed the same dose of 0.9% normal saline intragastrically every day for six weeks. All 36 rats tolerated the operation and were kept in single cages during the experiment.

Micro CT and angiography analyses
After general anesthesia with an intraperitoneal injection of 5% (w/v) chloral hydrate (0.8 ml/ 100 g), anteroposterior and lateral radiographs of the right hindlimb were obtained using Xray (50 kV and 4 mA; Kodak, Rochester, NY, USA) at 4 weeks after the operation. For the ECG, IMG, and IFG, the radiographs were obtained 6 weeks after the second-stage surgery. The radiographs were scored using the Lane-Sandhu scoring system. Scoring was carried out by three experts in the department of radiology.
For angiography, after anesthesia, the skin and subcutaneous tissues were incised under the xiphoid process of the sternum. The heart was exposed by an incision along the manubrium sternum and ribs. The perfusion needle was inserted from the right atrium to reach the aortic arch. The left atrial appendage was cut open, and 1% heparin saline was perfused until the fluid was clear and there was no blood outflow. After paraldehyde infusion and fixation, the Microfil (Microfil, MV-122, Flow Tech, Inc.) contrast agent was injected until it was completely drained (nearly 20 ml), put the animal in 4˚C overnight, and then collected the femur, decalcification thoroughly. Use the Micro-CT to scan and analysis.
For the Micro-CT scan, femur were collected, and the attached soft tissue was removed thoroughly and then fixed in 4% paraformaldehyde. The specimens were scanned with an animal micro-CT scanner (Skyscan 1172, Bruker, Madison, WI, USA). The Scanning parameters with an 80 kVp energy setting, intensity of 313 uA with 280 ms acquisition time and a image resolution of 120 μm. The defective region was identified by a contour as a traced region of interest (ROI).

Histoological analysis
Histological evaluations of the induced membranes were performed by microscope observations of stained tissue sections. Cryosections (5-μm thick) of the induced membrane were stained with hematoxylin and eosin (H&E) (Solaibao Technology Co., Ltd., Beijing, China). The components of the extracellular membrane were detected with an Elastica-van Gieson stain (Merck Serono KGaA, Darmstadt, Germany) and Muscovy O-solid green cartilage staining (Feijing Biotechnology Co., Ltd., Beijing, China), according to the manufacturers' instructions.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Pieces of membranes were immediately frozen after collection and stored in liquid nitrogen. Frozen membrane (200-600 mg) was powdered with a pestle and homogenized in TRIzol (Promega, Madison, WI, USA). RNA was used for cDNA synthesis with the RevertAid First-Strand Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) using oligo-dT as a primer. cDNA product (1 μl) was used for qRT-PCR with the SYBER Green technique (SYBR Green/ROX qPCR Master Mix) by quantifying the product at each cycle in a real-time manner. The amount of product was monitored for 40 cycles. The product specificity was confirmed by melting curve and, in some cases, by visualizing the products with agarose gel electrophoresis. The expression of β-actin was studied using the primer

Immunohistochemistry
Sections were obtained as mentioned above, and the slides were incubated overnight. The slides were washed for 15 min in 0.1% Triton X-100 diluted with PBS and blocked with 5%  SPSS 22.0 was used for statistical analysis. The continuous data were expressed as mean ± standard deviation (SD) and were analyzed using Student's t-test (two-group comparisons) or ANOVA with the LSD post hoc test (multiple group comparison). Categorical data were presented as n (%) and analyzed using the chi-square test. P-values <0.05 were considered statistically significant.

Examination of the bone defects and the Masquelet technique model
X-ray at 4 weeks after surgery showed that the alignment of the osteotomy fracture in the observation group was good. The plates and screws were in place, and the stable 4-mm bone defect area was maintained (Fig 1A). For the PMMA implantation, the cement was in a normal position without loosening or overturning (Fig 1B).
The induced membranes showed many small and round cells near the bone cement. A rich vascular network was formed far from the bone cement and adjacent to the muscle, while fibrous tissue parallel to the PMMA bone cement block was formed in the middle area ( Fig 1C).

RDTF improves ternary regulation related factors
Except for the EG group, the other groups had a more typical inductive membrane structure and more abundant vascular distribution (Fig 2A). To further explore the potential machanism of RDTF in Induced membrane formation, some related biomarkers were detected from protein and gene level respectively.
The immunohistochemistry results demonstrated that the expression of the CD31 protein in the EG group was weaker than that in the other groups (Fig 2B). The endomucin and PDGF-BB proteins were expressed in the induced membrane tissues of each group, but all of them were weakly positive. HMGB1 was expressed abundantly in the induced membrane tissue of each group.
PCR results showed that compared with the empty control group, the mRNA expression of CD31, VEGFA, HMGB1, and PDGF-BB in the IG, LG, MG, and HG were significantly increased. The expression of endomucin mRNA was not significantly increased, and the expression level was significantly increased only when with high-dose RDTF. After the use of RDTF, the ternary regulation-related factors VEGFA and HMGB1 tended to increase. After the use of RDTF, regardless of the dose, the expression of PDGF-BB mRNA was significantly increased compared with the IG group, while the expression of HMGB1 and endomucin significantly increased only when high doses of RDTF were used compared with those of IG. And VEGFA expression was significantly increased when the dose of RDTF reached the medium dose. The results of CD31 also showed a general trend of increased expression ( Fig  3A). Similar results were observed at the protein level (Fig 3B).

RDTF improves bone quality and blood vessels in the bone defect area
Six weeks after surgery, as presented in Fig 4, PMMA was radiopaque so that the bone substitute and the broken ends were visible. The defect in the IFG group showed continuous osteogenesis. Cortical bone was well reconstructed, and the marrow cavity had formed. The defect in the IMG group showed discontinuous osteogenesis. The bone marrow cavity was not fully formed, and the reconstructed bone cortex was weak, with discontinuous bone cortex still visible. Nothing grew in the critical-sized bone defects in the ECG group. The micro-CT crosssections showed more obvious new bone formation in the IFG group than in the other two groups (Fig 5).
In the ECG group, there were fibers, muscles, and connective tissues, but only small amounts of bone tissue, while the amount of bone tissue was higher in the IMG and IFG ( Fig  6A). IFG displayed the features of the osteoplastic stage, with homogeneous osteogenic distribution, while IMG displayed inflammatory cell infiltration, a small amount of proliferative connective tissue, and uneven osteogenesis.
Angiography showed that the blood vessels in the ECG group were dissected, and no obvious blood vessels were in the defect area. A small number of new blood vessels were observed in the defect area of the IMG, with continuous but uneven distribution. The quality of the blood vessels formed in the IFG was better, and the local blood supply was more abundant (Fig 6B).

Discussion
Rhizoma drynariae (gusuibu) is a classic herb used to treat fractures in traditional Chinese medicine [19,20], the main effective ingredient in Rhizoma drynariae is RDTF. CD31 hi Emcn hi vessels induced by PDGF-BB secreted by osteoclast precursors, together with osteoblasts and osteoclasts, constitute the ternary regulatory mechanism of bone tissue reconstruction [16]. This study aimed to determine whether RDTF can promote bone remodeling and induce membrane growth in the rat Masquelet model and to explore its molecular mechanism based on the ternary regulation theory. The preliminary results showed that RDTF positively affected angiogenesis and bone reconstruction in the bone defect area and on factors involved in ternary regulation.
The formation of the induction membrane is a foreign body reaction of tissues after PMMA implantation, which is embodied by inflammation and edema reaction induced by multinucleated giant cells [27]. At 2 weeks after implantation of PMMA, the induction membrane was observed to have internal and external structures. The inner layer is a synovium-like epithelial cell layer, and the outer layer is mainly composed of fibroblasts, myofibroblasts, and collagen [15,28]. Masquelet described the induced membrane as a 1 to 2mm thick and has a similar histologically characters to the synovial membrane, providing a microenvironment conducive to bone healing. It was also noted that the induction membrane could provide a very rich blood supply and prevent the soft tissues from growing into the bone defect area and the bone graft absorbed [29][30][31]. Induced membranes are formed in a complex biological environment in which multiple cytokine systems are involved [32][33][34]. As reported by Masquelet et al. [8] and Viateau et al. [35,36], the induced membrane is like periosteum, which both contain and promote regenerative bone cells, growth factors, and neovascularization. Pelissier et al. [9] showed large numbers of capillaries in the outer layer of the induced membrane tissue at various stages, with an average diameter of about 1.2 mm. Tang et al. [37] demonstrated that Dll4/Notch1 signaling is negatively associated with the vessel density of the induced membrane.
In the process of bone repair, the induction membrane acts as a bed for the bone graft and the role of recruiting and chemotactic BMSCs. Wang et al. [38] revealed that the MDSCs of the induced membrane promoted the angiogenesis of endothelial cells through the expression of VEGFA, Ang2, and HIF-1α, which was upregulated by the activation of STAT3 signaling. Due to the affinity between microvessels and osteoprogenitor cells and osteoblasts, the invasion of osteoprogenitor cells into the lesion area in the process of bone repair is often accompanied by vascular growth. There is growing evidence that new blood vessels do more than just establishing local circulation to the new bone and supplying oxygen and nutrients, and they can also directly promote osteogenesis [4]. The expression of neovascularization-related genes in the induced membrane tissue after intervention with RDTF could be used as the basis for future research. This study will also help better understand the biological properties of the induced membrane, which will help create a better microenvironment for bone healing.
This study showed that RDTF had a certain effect on the angiogenesis of the bone defect area, and the number of new vessels in the IFG group was significantly higher than the other groups. X-ray and micro-CT also showed that the mineralization was better than in the other two groups. It could promote the formation of callus in the defect area. From a macroscopical perspective, RDTF in the Masquelet technique positively affects angiogenesis and osteogenesis of the bone defect area.
CD31 expression in endothelial cells is closely related to endothelial cell migration and neovascularization and is commonly used as a marker to detect neovascularization [15]. Gouron et al. [15] confirmed that the formation of new blood vessels was continuously maintained during the whole formation process of the inducing membrane. Cuthbert et al. [39] described that CD31 was expressed in the vascular lumen edge in the immunohistochemical results, confirming the existence of abundant blood vessels in the induced membrane tissue. The immunohistochemistry results in this study confirmed the results of Cuthbert et al. [39], and CD31 was shown in the vascular lumen edge of the induced membrane tissue, which was consistent with the results of western blotting and qRT-PCR. Park-Windhol et al. [40] found that endomucin could regulate VEGFR2 activity and affect VEGF-induced endothelial cell migration, growth, and tube morphogenesis. This study suggests that endomucin is a potential regulator of angiogenesis and might provide new targeted therapies for vascular diseases. Different blood vessels could be identified by the immunohistochemistry of specific cells. Using this technique, Kusumbe et al. [41] found that endothelial cells in the endosteum were both strongly positive for CD31 and endomucin, while the venous sinus located in the diaphysis was only weakly positive for CD31 and weakly expressed endomucin. CD31 hi Emcn hi endothelial cells and CD31 lo Emcn lo cells were significantly different in the suspension cells of long bone [41]. Therefore, the authors first proposed a new term for intracapsular microvessels: CD31 hi Emcn hi vessels, also known as H-type vessels. Endomucin plays an important role in microangiogenesis, and if the immunohistochemistry results show strong CD31 and endomucin expression, the presence of H-type vessels may be suggested. The WB results in the present study confirmed the expression of PDGF-BB protein in the induced membrane tissue, so whether there were H-type blood vessels in the induced membrane tissue was explored. Still, the immunohistochemistry results showed that the expression of endomucin in the induced membrane was weak rather than strong positive, leading to the conclusion that there were no CD31 hi Emcn hi vessels in the induced membrane.
This study has limitations. The promoting effects of RDTF on each growth factor were not completely consistent, which might be related to the small sample size of this study and induced membrane sampling operation. The results showed that the concentration of RDTF had a certain effect on the secretion of each growth factor. In this study we didn't performed the experiment in vitro, in the further study we need to do it and to test more bio-markers, in this way we can have a better understanding the molecular mechanism of the ternary regulation theory.
In conclusion, the pathological, X-ray, western blot, and qRT-PCR results confirmed the secretion of VEGF, HMGB1, PDGF-BB, CD31, and endomucin in the induced membrane of the model group was significantly higher than that in the empty control group. Although the expected CD31 hi Emcn hi vessels in the induction membrane were not observed, this study confirmed that RDTF could promote the secretion of angiogenic factors in the induced membrane. The specific mechanisms still need to be further studied.